SCIENTIFIC RESEARCH PROJECT: SUMMARY Leukocytes are a functionally diverse group of cells that modulate the immune response and are involved in the induction and resolution of inflammation. BPH is associated with high levels of leukocytic infiltration representing both the lymphoid and myeloid lineages. To date our understanding of the leukocytes present in the prostate has been largely based upon analysis of the presence of cell surface marker combinations that define cell classes. The application of single cell (sc)RNA-seq to BPH patient samples in conjunction with bioinformatics analysis will provide a more comprehensive picture of the biological action of these cells. For example, our preliminary observations on changes in the CD68/CD163-expressing cell ratio with BPH progression are an incomplete picture of the changes that are occurring in macrophages present in BPH as the disease progresses. A more complete analysis of gene expression profiles in these cells will allow a deeper understanding of their actions and interactions with the stromal and epithelial cells within the prostate. Likewise our clinical observations relating to the effects of TNF? antagonists and their ability to suppress BPH incidence and progression would be complemented by more comprehensive data on the sources of this cytokine. Given the large number of new agents becoming available to target specific components of the immune/inflammatory network, the ability to start to track the interactions between the leukocytes in the prostate should also elucidate new therapeutic options. The central hypothesis of this proposal is that, in BPH patients there will be a change in the profile of gene expression in intraprostatic leukocytes as the prostate grows and BPH progresses. A detailed understanding of this process will inform the development of new therapeutic options helping to personalize BPH treatment. scRNA-seq will define specific subpopulations of leukocytes and the range of signaling molecules that they produce in BPH patients, elucidating chemokine/cytokine networks and identifying potential new therapeutic targets. We will generate a data set that will identify key leukocyte subpopulations and important pathways between them. These data will support future studies to investigate changes in the status of immune/inflammatory cells as BPH progresses. They will also be relevant to other causes of LUTS and benign urologic diseases with an inflammatory component, as well as to urologic malignancies. Two Specific Aims will be pursued by a multidisciplinary team. Define the profile of leukocytes and their changes during BPH progression based upon single-cell RNA-seq analysis. We will apply scRNA-seq to human BPH leukocytes to establish gene expression profiles in these cells and to identify the specific cell clusters present in the gland. Discover cellular clusters that possess distinct pathway topologies for key BPH pathways. We will apply heterogeneous Bayesian graphical models (HBGMs) to analyze scRNA-seq data to discover cell clusters that are estimated to have unique and different pathway topologies as sets of connected edges between genes and test these predicted changes in clinical samples.